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Specific activation of the cysteine protease CPP32 during the negative selection of T cells in the thymus.

Alam A, Braun MY, Hartgers F, Lesage S, Cohen L, Hugo P, Denis F, Sékaly RP - J. Exp. Med. (1997)

Bottom Line: This was observed both in anti-CD3- and dexamethasone-induced apoptosis.Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD.While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Immunologie, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7, Canada.

ABSTRACT
Cysteine proteases of the CED-3 and ICE family have been recently proposed as the ultimate executioners in several mammalian cell death pathways. Among them, the cysteine protease CPP32 has been shown to participate in programmed cell death (PCD), or apoptosis, affecting lymphoid cells in vitro. In the thymus, negative selection is a mechanism through which developing thymocytes expressing a TcR with high affinity for self peptide-MHC complexes are eliminated by PCD. In order to investigate the role of CPP32 in thymic apoptosis, isolated thymocytes were submitted to cell surface CD3 crosslinking by immobilized anti-CD3 mAb or to dexamethasone treatment. Although apoptosis occurred in the absence or after crosslinking with anti-CD3 mAb, specific activation of CPP32, as assessed by the extent of proteolytic cleavage of the p32 zymogen, was only detected in thymocytes cultured in the presence of the immobilized antibody or dexamethasone. This activation was a very early event during apoptosis as it occurred before the exposure of phosphatidyl serine to the upper side of the cell membrane. This was observed both in anti-CD3- and dexamethasone-induced apoptosis. Moreover, using mice transgenic for pigeon cytochrome C (PCC)-specific TcR, we were able to show that, after injection of PCC, the activation of CPP32 and cleavage of its substrate occurred in thymocytes obtained from mice expressing a permissive MHC haplotype for PCC presentation (H-2k). Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD. While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F. Taken together, these results support the concept that CPP32 is among the earliest effectors of the pathway leading to negative selection of autoreactive thymocytes. Our results also suggest the involvement of a distinct CPP32-like cysteine protease in spontaneous apoptosis of thymocytes.

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Injection of PCC induce negative selection and activation of CPP32 in thymocytes of PCC-specific TcR transgenic mice of the H-2k, but  not H-2b, haplotype. (A) Absolute number of cells in the different cell populations of thymuses isolated from H-2k and H-2b TcR transgenic mice 24 h  after PCC injection. (B, top) Percentages of expression of CD4 and CD8 molecules by viable thymocytes (annexin V−) isolated from PCC-injected  H-2k and H-2b TcR transgenic mice after 18 h of culture at 37°C. Thymuses from noninjected H-2k TcR transgenic mice contain 34.3 ± 2.5% of  CD4+8− cells, 48.4 ± 5.7% of CD4+8+ cells and 15.0 ± 5.6% of CD4−8− cells. Thymuses from noninjected H-2b TcR transgenic mice contain 37.0 ±  1.8% of CD4+8− cells, 46.0 ± 4.1% of CD4+8+ cells and 14.8 ± 3.9% of CD4−8− cells. (B, bottom) Percentages of annexin V+ cells in CD4+8−,  CD4+8+ and CD4−8− thymocytes isolated from PCC-injected H-2k and H-2b TcR transgenic mice after 18 h of culture at 37°C. (C) Proteolytic cleavage of CPP32 and PARP in thymocytes isolated from PCC-injected H-2k and H-2b TcR transgenic mice. After 6 or 18 h of culture at 37°C, cells were  lysed and the lysates were electrophoresed. The substrate cleavage was visualized by immunoblotting as described in Materials and Methods.
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Figure 6: Injection of PCC induce negative selection and activation of CPP32 in thymocytes of PCC-specific TcR transgenic mice of the H-2k, but not H-2b, haplotype. (A) Absolute number of cells in the different cell populations of thymuses isolated from H-2k and H-2b TcR transgenic mice 24 h after PCC injection. (B, top) Percentages of expression of CD4 and CD8 molecules by viable thymocytes (annexin V−) isolated from PCC-injected H-2k and H-2b TcR transgenic mice after 18 h of culture at 37°C. Thymuses from noninjected H-2k TcR transgenic mice contain 34.3 ± 2.5% of CD4+8− cells, 48.4 ± 5.7% of CD4+8+ cells and 15.0 ± 5.6% of CD4−8− cells. Thymuses from noninjected H-2b TcR transgenic mice contain 37.0 ± 1.8% of CD4+8− cells, 46.0 ± 4.1% of CD4+8+ cells and 14.8 ± 3.9% of CD4−8− cells. (B, bottom) Percentages of annexin V+ cells in CD4+8−, CD4+8+ and CD4−8− thymocytes isolated from PCC-injected H-2k and H-2b TcR transgenic mice after 18 h of culture at 37°C. (C) Proteolytic cleavage of CPP32 and PARP in thymocytes isolated from PCC-injected H-2k and H-2b TcR transgenic mice. After 6 or 18 h of culture at 37°C, cells were lysed and the lysates were electrophoresed. The substrate cleavage was visualized by immunoblotting as described in Materials and Methods.

Mentions: Exposure to CD3-specific antibodies, in vitro and in vivo, stimulates apoptosis predominantly in CD4+8+ thymocytes (26–30). Based on the above observations it was tempting to suggest that, in vivo, CPP32 could take an active part in TcR-induced negative selection of autoreactive CD4+8+ T cells in the thymus. In order to verify this hypothesis, mice expressing an IEk-restricted pigeon cytochrome C (PCC)-specific TcR transgene (AD10) were injected intraperitoneally with PCC. 24 h after PCC challenge, the expression of the early activation marker CD69 on isolated thymocytes was only observed in PCC-injected AD10 mice of the H-2k and not of the H-2b haplotype (data not shown). Moreover, the thymus cellularity in injected H-2k animals was greatly affected (Fig. 6 A). The absolute number of CD4+8+ cells was at least twice lower in H-2k mice than in H-2b mice. As the absolute number of CD4+8− cells did not differ significantly between the two types of animals, the drop of CD4+8+ cell number in H-2k mice was interpreted as the result of PCC-stimulated PCD rather than of PCC-induced maturation in CD4+8− cells. Altogether these observations indicated that H-2k but not H-2b thymocytes were stimulated in vivo by PCC and were undergoing negative selection. Interestingly, the number of CD4−8− cells was always two fold higher in H-2k– injected mice as compared with H-2b animals. It is known that, soon after expressing a functional preTcR, CD4−8− thymocytes undergo a step of proliferation leading to maturation into the CD4+8+ stage (31). As in these two types of mice immature CD4−8− thymocytes express the TcR transgene, it is possible that injection of PCC in H-2k mice stimulates these cells to proliferate.


Specific activation of the cysteine protease CPP32 during the negative selection of T cells in the thymus.

Alam A, Braun MY, Hartgers F, Lesage S, Cohen L, Hugo P, Denis F, Sékaly RP - J. Exp. Med. (1997)

Injection of PCC induce negative selection and activation of CPP32 in thymocytes of PCC-specific TcR transgenic mice of the H-2k, but  not H-2b, haplotype. (A) Absolute number of cells in the different cell populations of thymuses isolated from H-2k and H-2b TcR transgenic mice 24 h  after PCC injection. (B, top) Percentages of expression of CD4 and CD8 molecules by viable thymocytes (annexin V−) isolated from PCC-injected  H-2k and H-2b TcR transgenic mice after 18 h of culture at 37°C. Thymuses from noninjected H-2k TcR transgenic mice contain 34.3 ± 2.5% of  CD4+8− cells, 48.4 ± 5.7% of CD4+8+ cells and 15.0 ± 5.6% of CD4−8− cells. Thymuses from noninjected H-2b TcR transgenic mice contain 37.0 ±  1.8% of CD4+8− cells, 46.0 ± 4.1% of CD4+8+ cells and 14.8 ± 3.9% of CD4−8− cells. (B, bottom) Percentages of annexin V+ cells in CD4+8−,  CD4+8+ and CD4−8− thymocytes isolated from PCC-injected H-2k and H-2b TcR transgenic mice after 18 h of culture at 37°C. (C) Proteolytic cleavage of CPP32 and PARP in thymocytes isolated from PCC-injected H-2k and H-2b TcR transgenic mice. After 6 or 18 h of culture at 37°C, cells were  lysed and the lysates were electrophoresed. The substrate cleavage was visualized by immunoblotting as described in Materials and Methods.
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Figure 6: Injection of PCC induce negative selection and activation of CPP32 in thymocytes of PCC-specific TcR transgenic mice of the H-2k, but not H-2b, haplotype. (A) Absolute number of cells in the different cell populations of thymuses isolated from H-2k and H-2b TcR transgenic mice 24 h after PCC injection. (B, top) Percentages of expression of CD4 and CD8 molecules by viable thymocytes (annexin V−) isolated from PCC-injected H-2k and H-2b TcR transgenic mice after 18 h of culture at 37°C. Thymuses from noninjected H-2k TcR transgenic mice contain 34.3 ± 2.5% of CD4+8− cells, 48.4 ± 5.7% of CD4+8+ cells and 15.0 ± 5.6% of CD4−8− cells. Thymuses from noninjected H-2b TcR transgenic mice contain 37.0 ± 1.8% of CD4+8− cells, 46.0 ± 4.1% of CD4+8+ cells and 14.8 ± 3.9% of CD4−8− cells. (B, bottom) Percentages of annexin V+ cells in CD4+8−, CD4+8+ and CD4−8− thymocytes isolated from PCC-injected H-2k and H-2b TcR transgenic mice after 18 h of culture at 37°C. (C) Proteolytic cleavage of CPP32 and PARP in thymocytes isolated from PCC-injected H-2k and H-2b TcR transgenic mice. After 6 or 18 h of culture at 37°C, cells were lysed and the lysates were electrophoresed. The substrate cleavage was visualized by immunoblotting as described in Materials and Methods.
Mentions: Exposure to CD3-specific antibodies, in vitro and in vivo, stimulates apoptosis predominantly in CD4+8+ thymocytes (26–30). Based on the above observations it was tempting to suggest that, in vivo, CPP32 could take an active part in TcR-induced negative selection of autoreactive CD4+8+ T cells in the thymus. In order to verify this hypothesis, mice expressing an IEk-restricted pigeon cytochrome C (PCC)-specific TcR transgene (AD10) were injected intraperitoneally with PCC. 24 h after PCC challenge, the expression of the early activation marker CD69 on isolated thymocytes was only observed in PCC-injected AD10 mice of the H-2k and not of the H-2b haplotype (data not shown). Moreover, the thymus cellularity in injected H-2k animals was greatly affected (Fig. 6 A). The absolute number of CD4+8+ cells was at least twice lower in H-2k mice than in H-2b mice. As the absolute number of CD4+8− cells did not differ significantly between the two types of animals, the drop of CD4+8+ cell number in H-2k mice was interpreted as the result of PCC-stimulated PCD rather than of PCC-induced maturation in CD4+8− cells. Altogether these observations indicated that H-2k but not H-2b thymocytes were stimulated in vivo by PCC and were undergoing negative selection. Interestingly, the number of CD4−8− cells was always two fold higher in H-2k– injected mice as compared with H-2b animals. It is known that, soon after expressing a functional preTcR, CD4−8− thymocytes undergo a step of proliferation leading to maturation into the CD4+8+ stage (31). As in these two types of mice immature CD4−8− thymocytes express the TcR transgene, it is possible that injection of PCC in H-2k mice stimulates these cells to proliferate.

Bottom Line: This was observed both in anti-CD3- and dexamethasone-induced apoptosis.Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD.While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Immunologie, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7, Canada.

ABSTRACT
Cysteine proteases of the CED-3 and ICE family have been recently proposed as the ultimate executioners in several mammalian cell death pathways. Among them, the cysteine protease CPP32 has been shown to participate in programmed cell death (PCD), or apoptosis, affecting lymphoid cells in vitro. In the thymus, negative selection is a mechanism through which developing thymocytes expressing a TcR with high affinity for self peptide-MHC complexes are eliminated by PCD. In order to investigate the role of CPP32 in thymic apoptosis, isolated thymocytes were submitted to cell surface CD3 crosslinking by immobilized anti-CD3 mAb or to dexamethasone treatment. Although apoptosis occurred in the absence or after crosslinking with anti-CD3 mAb, specific activation of CPP32, as assessed by the extent of proteolytic cleavage of the p32 zymogen, was only detected in thymocytes cultured in the presence of the immobilized antibody or dexamethasone. This activation was a very early event during apoptosis as it occurred before the exposure of phosphatidyl serine to the upper side of the cell membrane. This was observed both in anti-CD3- and dexamethasone-induced apoptosis. Moreover, using mice transgenic for pigeon cytochrome C (PCC)-specific TcR, we were able to show that, after injection of PCC, the activation of CPP32 and cleavage of its substrate occurred in thymocytes obtained from mice expressing a permissive MHC haplotype for PCC presentation (H-2k). Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD. While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F. Taken together, these results support the concept that CPP32 is among the earliest effectors of the pathway leading to negative selection of autoreactive thymocytes. Our results also suggest the involvement of a distinct CPP32-like cysteine protease in spontaneous apoptosis of thymocytes.

Show MeSH
Related in: MedlinePlus